5-HT1D and 5-HT1E/1F Binding Sites in Depressed Suicides
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Molecular Psychiatry (1997) 2, 314–321 1997 Stockton Press All rights reserved 1359–4184/97 $12.00 ORIGINAL RESEARCH ARTICLE 5-HT1D and 5-HT1E/1F binding sites in depressed suicides: increased 5-HT1D binding in globus pallidus but not cortex S Lowther1, CLE Katona2, MR Crompton3 and RW Horton1 1Department of Pharmacology and Clinical Pharmacology; 3Department of Forensic Medicine, St George’s Hospital Medical School, London SW17 0RE; 2Department of Psychiatry, University College London Medical School, London W1N 8AA, UK 5-HT1D and 5-HT1E/1F receptor binding sites were measured in brain samples obtained at post- mortem from suicide victims with a firm retrospective diagnosis of depression, and matched controls. In antidepressant-free suicides a significantly higher number of 5-HT1D receptors was found in globus pallidus. This was largely restricted to those suicides who died by violent means. This effect was not observed in antidepressant-treated suicides. No differences or trends in 5-HT1D binding were found in putamen, parietal or frontal cortex, in antidepressant- free or antidepressant-treated suicides. There were no differences in the number of 5-HT1E/1F receptors in any of the regions studied. Keywords: 5-HT1D receptor binding sites; 5-HT1E/1F receptor binding sites; suicide; depression; post- mortem brain tissue; antidepressant drugs; cerebral cortex; putamen; globus pallidus; basal ganglia Introduction receptors in a subgroup of subjects with no evidence of depressive symptoms, but not in a smaller subgroup There is abundant evidence that serotonergic function with well documented evidence of depression. is reduced in depression.1,2 Furthermore a consistent A number of 5-HT1D ligands and radioligands have feature of effective treatments is their ability to 10–13 been developed. The 5-HT a and 5-HT b recep- enhance serotonergic transmission.3 1D 1D tors have a very similar pharmacology, but can be dis- Seven classes of 5-HT (5-hydroxytryptamine, tinguished using ketanserin and ritanserin.14 [125I]GTI, serotonin) receptors are recognised; 5-HT have been 1–4 [3H]L-694,247 and [3H]GR 125743 are selective for 5- pharmacologically characterised, 5-HT were ident- 5–7 HT receptors,10,11,15–17 but do not distinguish the two ified by molecular biology and are less well character- 1D forms of the receptor. The 5-HT b receptor appears to ised.4 The 5-HT family is the largest with five subtypes 1D 1 be the dominant 5-HT receptor in human brain. termed 5-HT , 5-HT , 5-HT , 5-HT and 5-HT (the 1D 1A 1B 1D 1E 1F There are no selective ligands for 5-HT and 5-HT 5-HT receptor is now accepted as belonging to the 5- 1E 1F 1C receptors. They are labelled by [3H]5-HT and are HT family, and is termed 5-HT ).4 Two types of 5- 2 2C characterised by their low affinity for 5-carboxamido- HT1D receptor have been reported, termed 5-HT1Da and 5,6 tryptamine (5-CT), compared to other 5-HT1 recep- 5-HT b. The 5-HT receptor in the rat and mouse 1D 1B tors.18,19 In human frontal cortex these receptors is the species homologue of the 5-HT b receptor in 1D account for over 40% of the total 5-HT receptors.20 humans. 1 They have not been studied to date in relation to Studies of the possible involvement of 5-HT recep- 1 depression. tors in depression have been largely limited to the 5- In the present study we have used [3H]5-HT with HT receptor. The 5-HT receptor is of potential 1A 1D selective pharmacological blockade to examine 5-HT interest because it functions as a nerve terminal autore- 1D and 5-HT receptors (see Methods) in a group of ceptor, regulating the release of 5-HT.7,8 To date only 1E/1F suicides, restricted to those with a firm retrospective one study has examined 5-HT receptors in relation 1D diagnosis of depression. We have studied separately to depression. Arranz et al9 measured 5-HT receptor 1D those subjects who were free of antidepressants for at binding in frontal cortex of suicides. Although they least 3 months prior to death, and those in whom the found no significant difference overall compared to prescription of antidepressant drugs was clearly docu- matched controls, they found lower numbers of 5-HT 1D mented. Four brain regions were studied — frontal and parietal cortex, putamen and globus pallidus. We chose frontal cortex as this region is implicated in depression Correspondence: S Lowther, Department of Pharmacology and 21 Clinical Pharmacology, St George’s Hospital Medical School, and receptor changes have been reported. Another London SW17 0RE, UK. E-mail: slowtherKsghms.ac.uk cortical region, the parietal cortex, which has not been Received 10 October 1996; revised and accepted 23 January 1997 particularly implicated in depression, was chosen for 5-HT1D binding in depressed suicides S Lowther et al 315 comparative purposes. We have selected two areas of oughly mixed and stored in air-tight containers at the basal ganglia for study since structural and meta- −80°C until assayed. Brain areas studied were frontal bolic changes have been reported in affective dis- cortex (Brodmann area 10), parietal cortex (Brodmann orders.22–24 In human brain, the highest density of 5- area 7), putamen and globus pallidus. 20 HT1D receptors is found in globus pallidus, making it potentially interesting in relation to depression. Tissue preparation Membranes were prepared essentially as described by Cheetham et al.26 Frozen brain tissue was homogenised Materials and methods in ice-cold 0.25 M sucrose (1 : 20 w/v) using a motor- Subject selection driven teflon pestle (eight strokes at 120 rpm), and cen- Deaths recorded as suicide at Coroners’ inquest were trifuged (at 4°C) at 1000 g for 10 min. The supernatant subjected to retrospective diagnosis by a psychiatrist was stored on ice and the pellet rehomogenised in (CLEK), using hospital and Coroners’ records and inter- 0.25 M sucrose (1 : 10 w/v) and centrifuged at 750 g for views with the subject’s medical practitioner. Suicides 10 min. Combined supernatants were diluted with were divided into five groups according to the classi- 50 mM Tris-HCl buffer, pH 7.4 (at 25°C) to 1 : 100 w/v fication of Beskow et al.25 The subjects included in this and centrifuged at 35 000 g for 10 min. The membrane study came from group 1 (endogenous depression) and pellet was resuspended in 50 mM Tris-HCl buffer group 2 (depressive syndrome) only. Subjects from (1 : 50 w/v) and incubated at 37°C for 10 min to remove groups 3, 4 and 5, with diagnoses of schizophrenia, endogenous 5-HT, then centrifuged at 35 000 g for 10 personality disorder, epilepsy, alcohol or other drug min. The pellet was washed by resuspension in 50 mM abuse, alone or in combination with depression, and Tris-HCl buffer (1 : 100 w/v) and centrifuged at those with no clear psychiatric diagnosis, were 35 000 g for 10 min. The final pellet was resuspended excluded. Information on current and previous drug in 50 mM Tris-HCl buffer at a concentration of 5.3 mg treatment was sought and blood samples obtained at original wet weight ml−1 for cortical areas and post-mortem were analysed for the presence of psy- 2.6 mg ml−1 for putamen and globus pallidus, and choactive drugs. This information allowed two distinct immediately used in the binding assay. groups of suicides to be studied. Group 1. Antidepressant-free suicides (n = 20). These Binding assays subjects had not been prescribed antidepressant drugs Total 5-HT1, 5-HT1D/1E/1F and 5-HT1E/1F binding was within 3 months prior to death. Causes of death were determined concurrently (as previously described).20 hanging (n = 8), carbon monoxide poisoning (n = 5), Assays were performed in 5-ml plastic tubes. Drug sol- drug overdosage (n = 5), self-inflicted wounding (n = 1) utions were prepared in water, and ligand in 50 mM and jumping from height (n = 1). Control brains for this Tris-HCl buffer, pH 7.4 (at 25°C). Common to all assays group were from subjects who died suddenly from were 500 ml freshly prepared membranes, 100 mlof causes not involving the central nervous system, and 50 mM Tris-HCl buffer, pH 7.4 containing 4 mM cal- without documented evidence of mental illness. Con- cium chloride (final concentration) and 0.1% ascorb- trols were individually matched for age (75% within ate, 100 ml water (total binding) or unlabelled 5-HT 5 years) and gender. Causes of death were myocardial (10 mM, to define non-specific binding) and 100 ml infarction (n = 17), accidental fall (n = 1), acute asthma [3H]5-HT (25.4 Ci mmol−1, NEN Dupont, Boston, MA, (n = 1) and road traffic accident (n = 1). USA) at eight concentrations (0.5–32 nM final). 5- = Group 2. Antidepressant-treated suicides (n 16). HT1D/1E/1F binding sites were characterised by the pres- Prescription of antidepressant drugs was clearly docu- ence of 100 ml(−)pindolol (500 nM final) and 100 ml mented in this group. Details of drug treatment and mesulergine (100 nM final) to block 5-HT1A and 5-HT2C causes of death for these suicides are shown in Table 1. receptors respectively. (5-HT1B receptors are absent in 27 A second control group was individually matched for human brain.) 5-HT1E/1F binding was characterised by age (100% within 5 years) and gender to the antide- 100 ml 5-carboxamidotryptamine (5-CT, 100 nM final) m pressant-treated suicides.